Striatal-enriched protein tyrosine phosphatase expression and activity in Huntington's disease: a STEP in the resistance to excitotoxicity

Abstract

Striatal-enriched protein tyrosine phosphatase (STEP) is highly expressed in striatal projection neurons, the neuronal population most affected in Huntington's disease. Here, we examined STEP expression and phosphorylation, which regulates its activity, in N-terminal exon-1 and full-length mutant huntingtin mouse models. R6/1 mice displayed reduced STEP protein levels in the striatum and cortex, whereas its phosphorylation was increased in the striatum, cortex, and hippocampus. The early increase in striatal STEP phosphorylation levels correlated with a deregulation of the protein kinase A pathway, and decreased calcineurin activity at later stages further contributes to an enhancement of STEP phosphorylation and inactivation. Accordingly, we detected an accumulation of phosphorylated ERK2 and p38, two targets of STEP, in R6/1 mice striatum at advanced stages of the disease. Activation of STEP participates in excitotoxic-induced cell death. Because Huntington's disease mouse models develop resistance to excitotoxicity, we analyzed whether decreased STEP activity was involved in this process. After intrastriatal quinolinic acid (QUIN) injection, we detected higher phosphorylated STEP levels in R6/1 than in wild-type mice, suggesting that STEP inactivation could mediate neuroprotection in R6/1 striatum. In agreement, intrastriatal injection of TAT-STEP increased QUIN-induced cell death. R6/2, Tet/HD94, and Hdh(Q7/Q111) mice striatum also displayed decreased STEP protein and increased phosphorylation levels. In Tet/HD94 mice striatum, mutant huntingtin transgene shutdown reestablished STEP expression. In conclusion, the STEP pathway is severely downregulated in the presence of mutant huntingtin and may participate in compensatory mechanisms activated by striatal neurons that lead to resistance to excitotoxicity.

Figure 1.

STEP protein and mRNA levels are decreased in the striatum of R6/1 mice. STEP protein levels (A, B) were analyzed by Western blot of protein extracts obtained from the striatum of WT and R6/1 mice at different stages of the disease progression (from 4 to 30 weeks of age). A, B, Representative immunoblots show the protein levels of STEP₆₁, STEP₄₆, and α-tubulin in WT and R6/1 mice at 4, 8, and 30 weeks of age. The graphs show the decrease in striatal STEP₆₁ (A) and STEP₄₆ (B) protein levels in R6/1 mice with respect to their littermate controls at different stages of the disease progression. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of WT mice (STEP₆₁ or STEP₄₆/α-tubulin ratio) and shown as mean ± SEM (n = 9). C, Graph showing STEP mRNA levels analyzed by Q-PCR in the striatum of 8- and 20-week-old WT and R6/1 mice. Results were normalized to the 18S gene expression, expressed as percentage of WT values, and shown as mean ± SEM (n = 5–7 for each genotype). Data were analyzed by Student's t test. **p < 0.01 and ***p < 0.001 compared with WT mice.

Figure 2.

A, B, STEP phosphorylation is increased in the striatum of R6/1 mice. pSTEP₆₁ (A) and pSTEP₄₆ (B) levels were analyzed by Western blot of protein extracts obtained from the striatum of WT and R6/1 mice at different stages of the disease progression (from 4 to 30 weeks of age). Representative immunoblots show protein levels of pSTEP₆₁, pSTEP₄₆, STEP₆₁, STEP₄₆, and α-tubulin in WT and R6/1 mice at 4, 8, and 30 weeks of age. The graphs show striatal pSTEP levels in R6/1 mice with respect to their littermate controls at different stages of the disease progression. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of age-matched WT mice (pSTEP₆₁/STEP₆₁ or pSTEP₄₆/STEP₄₆ ratio after normalization with α-tubulin) and shown as mean ± SEM (n = 4–7). Data were analyzed by Student's t test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with WT mice; ^##p < 0.01 and ^###p < 0.001 compared with 12-week-old R6/1 mice; and ⁺p < 0.05 compared with 20-week-old R6/1 mice.

Figure 3.

pERK2 and p-p38 levels are increased in the striatum of R6/1 mice. pERK2 (A) and p-p38 (B) levels were analyzed by Western blot of protein extracts obtained from the striatum of 8- to 30-week-old WT and R6/1 mice. Representative immunoblots show protein levels of pERK2, ERK2, p-p38, p38, and α-tubulin in WT and R6/1 mice at 8, 20, and 30 weeks of age. The graphs show that the striatal levels of pERK2 and p-p38 are significantly increased in R6/1 mice with respect to their littermate controls at 20 and 30 weeks of age. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of age-matched WT mice (pERK2/ERK2 and p-p38/p38 ratio after normalization with α-tubulin) and shown as mean ± SEM (n = 6). Data were analyzed by Student's t test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with WT mice.

Figure 4.

Regulation of NR1, DARPP-32, and STEP phosphorylation in the striatum. A, B, The phosphorylation levels of a specific PKA residue on NR1 (Ser897) (A) and on DARPP-32 (Thr34) (B) were analyzed by Western blot of protein extracts obtained from the striatum of WT and R6/1 mice at 4, 12, and 30 weeks of age. Representative immunoblots are presented. The graphs show increased levels of pNR1 (Ser897) (A) and pDARPP-32 (Thr34) (B) in R6/1 mice with respect to their littermate controls at different stages of the disease progression. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of age-matched WT mice [pNR1 (Ser897)/NR1 ratio or pDARPP-32 (Thr34)/DARPP-32 ratio after normalization with α-tubulin] and shown as mean ± SEM (n = 8). Data were analyzed by Student's t test. *p < 0.05 and **p < 0.01 compared with WT mice; and ^#p < 0.05 compared with 12-week-old R6/1 mice. C, PKA activation or calcineurin inhibition increases pSTEP₆₁ levels in the striatum. Twelve-week-old WT mice (n = 6 for each condition) received an intraperitoneal injection of vehicle, papaverine (30 mg/kg; Papav.), or FK-506 (5 mg/kg), and striatal pSTEP₆₁ levels were analyzed by Western blot (10 min after papaverine and 2.5 h after FK-506 injection). Representative immunoblots show protein levels of pSTEP₆₁, STEP₆₁, and α-tubulin in all conditions examined. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of vehicle-injected mice (pSTEP₆₁/STEP₆₁ ratio after normalization with α-tubulin) and shown as mean ± SEM. D, PKA activation, but not calcineurin inhibition, increases pNR1 (Ser897) levels in the striatum. Twelve-week-old WT mice (n = 6 for each condition) received an intraperitoneal injection of vehicle, papaverine (30 mg/kg; Papav.), or FK-506 (5 mg/kg), and striatal pNR1 (Ser897) levels were analyzed by Western blot. Representative immunoblots show protein levels of pNR1 (Ser897) and α-tubulin in all conditions examined. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of vehicle-injected mice [pNR1 (Ser897)/α-tubulin ratio] and shown as mean ± SEM. Data were analyzed by Student's t test. **p < 0.01 and ***p < 0.001 compared with vehicle-injected mice.

Figure 5.

A, B, Regulation of STEP and ERK2 signaling after intrastriatal QUIN injection. pSTEP₆₁ (A) pSTEP₄₆ (B), and pERK2 (C) levels were analyzed by Western blot of protein extracts obtained from the striatum of 12-week-old WT and R6/1 mice, 1 and 4 h after an intrastriatal injection of vehicle or QUIN (10 nmol). Representative immunoblots show protein levels of pSTEP₆₁, STEP₆₁, and α-tubulin (A), pSTEP₄₆, STEP₄₆, and α-tubulin (B), and pERK2, ERK2, and α-tubulin (C) in WT and R6/1 striatum, 1 and 4 h after QUIN injection. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of the contralateral vehicle-injected WT striatum (pSTEP₆₁/STEP₆₁ ratio, pSTEP₄₆/STEP₄₆ ratio, or pERK2/ERK2 ratio, after normalization with α-tubulin), and data shown are the mean ± SEM (n = 7–9). Data were analyzed by two-way ANOVA, followed by Bonferroni's post hoc test. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with vehicle-injected contralateral WT striatum. WT, Vehicle-injected WT striatum; WT + QUIN, QUIN-injected WT striatum; R6/1, vehicle-injected R6/1 striatum; R6/1 + QUIN, QUIN-injected R6/1 striatum.

Figure 6.

Inhibition of PDE10A or calcineurin increases pERK2 levels in the striatum. Twelve-week-old wild-type mice (n = 6 for each condition) received an intraperitoneal injection of vehicle, papaverine (30 mg/kg; Papav.), or FK-506 (5 mg/kg), and striatal pERK2 levels were analyzed by Western blot (10 min after papaverine and 2.5 h after FK-506 injection). Representative immunoblots show protein levels of pERK2, ERK2, and α-tubulin in all the conditions examined. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of vehicle-injected mice (pERK2/ERK2 ratio after normalization with α-tubulin) and expressed as mean ± SEM. Data were analyzed by Student's t test. *p < 0.05 and **p < 0.01 compared with vehicle-injected mice.

Figure 7.

Intrastriatal TAT–STEP injection increases QUIN-induced cell death in R6/1 mice striatum. Control peptide (TAT–myc) or TAT–STEP was intrastriatally injected in R6/1 mice 1 h before intrastriatal QUIN injection. A, Cell death was assessed by Fluoro-Jade staining 24 h after QUIN injection. Representative photomicrographs show the striatal area occupied by Fluoro-Jade-positive cells in R6/1 mice striatum injected with QUIN (a1), TAT–myc plus QUIN (a2), or TAT–STEP plus QUIN (a3). B, Immunohistochemical staining against pERK2 was performed 24 h after QUIN injection in R6/1 mice striatum with or without previous injection of TAT–STEP. Representative images showing the striatum in all the conditions analyzed. In the inset, note the distinct pERK2 immunoreactivity in non-injured (1) and injured (2) striatal cells. Scale bar, 500 μm.

Figure 8.

Alterations of STEP and pSTEP levels in the striatum of R6/2 and Hdh^Q7/Q111 mice. A, B, pSTEP₆₁, pSTEP₄₆, STEP₆₁, and STEP₄₆ levels were analyzed by Western blot of protein extracts obtained from the striatum of WT and R6/2 mice at 12 weeks of age (A) and from 8-month-old Hdh^Q7/Q7 and knock-in Hdh^Q7/Q111 mice (B). Representative immunoblots show the protein levels of pSTEP₆₁, pSTEP_46, STEP₆₁, STEP₄₆, and α-tubulin in WT, Hdh^Q7/Q7, R6/2, and Hdh^Q7/Q111 mice. Values (obtained by densitometric analysis of Western blot data) are expressed as percentage of WT/Hdh^Q7/Q7 mice (pSTEP/STEP ratio after normalization with α-tubulin and STEP/α-tubulin ratio, respectively) and shown as mean ± SEM (n = 5–8). Data were analyzed by Student's t test. *p < 0.05 and ***p < 0.001 compared with WT or Hdh^Q7/Q7 mice, respectively.

Figure 9.

Changes in STEP expression but not in STEP phosphorylation are reverted by suppressing the mhtt transgene expression in Tet/HD94 mice striatum. pSTEP₆₁, STEP₆₁, and STEP₄₆ protein levels were analyzed by Western blot in the striatum of 22-month-old WT and Tet/HD94 mice, either with no pharmacological intervention (Gene-ON) or after 5 months of mhtt transgene shutdown by doxycycline administration (Gene-OFF). A, B, The graphs show the densitometric measures of STEP₆₁ and STEP₄₆ normalized to α-tubulin (A) and the pSTEP₆₁/STEP ratio after normalization with α-tubulin (B). Results are expressed as percentages of WT ± SEM. Data were analyzed by one-way ANOVA, followed by Bonferroni's post hoc test. *p < 0.05 compared with WT mice; ^#p < 0.05 compared with Tet/HD94 gene-ON mice.